Additional file 1.
Graphic representations of recombinant constructs, mammalian plasmid vector, and single LASV gene expression. Ai. GPC gene with known domains (SP, signal peptide; GP1, glycoprotein 1; GP2, glycoprotein 2; TM, transmembrane; IC, intracellular; ER, endoplasmic reticulum retention signal). Signal peptidase (SPase) and subtilase SKI-1/S1P cleavage sites are indicated. Seven glycosylation sites on GP1 and 4 on GP2 are indicated by Y. Aii. GPC construct with C-terminal FLAG. Aiii. Nucleoprotein gene displaying putative helicase, RNA binding, WD40, repeated [R] domains, and pre-protein cleavage motif. Aiv. NP with C-terminal 6X-HIS. Av. Z gene displaying myristoylation (myr), cyclin/CDK, nuclear receptor box (NR BOX), RING, and late PTAP and PPPY domains. Avi. Z gene with one glycine-6X-HIS domain inserted at amino acid position +3. Avii. Z gene with C-terminal 6X-HIS. B. Mammalian expression vector pcDNA3.1+_intA was used to generate all expression constructs outlined in these studies. C. LASV NP-3'HIS (lane 1), Z-3'HIS (lane 2), Z-5'glyHIS (lane 3), and GPC (lane 4) gene expression were analyzed by western blot. Ci. Intracellular (C) expression of NP-3'HIS (60 kDa), Z-3'HIS (12 kDa), Z-5'glyHIS (15 kDa), and GPC (72 kDa). In the GPC lane, probed with an α-GP1 mAb, expression of monomeric GP1 was also detected (42 kDa). In culture supernatants (S), NP-3'HIS was not detected (Cii, lane 1). Z-3'HIS was present in supernatants at high levels (Cii, lane 2). Disrupting the myristoylation site on the N-terminus of Z prevented the release of the protein from cells (Cii, lane 3). The soluble GP1 component previously described through expression of GPC [11,12] was detected in supernatants (42 kDa) (Cii, lane 4).
Format: PPTX Size: 145KB Download file
Branco et al. Virology Journal 2010 7:279 doi:10.1186/1743-422X-7-279